Insect baculoviruses are still the potential group of insect pathogenic viruses to be used as biological control agents. Agricultural pests are to a large extent created by human interference and are a consequence of imbalance in the natural equilibrium. Our usual way cf controlling pests with chemicals has created more problems than it has solved. For the future we have to accept the fact that the, preservation of the natural environment as a whole is at least as important as the protection of a spiecific crop. So the application of naturally occurring agents to control pests and to restore the natural equilibrium appears to be an obvious approach.
A plaque assay and cloning procedure a ere developed for the baculovirus, Autographs californica nuclear polyhedrosis virus (AcNPV). Twelve nondefective clones were isolated using extracellnlar nonoccluded virions which were shown to contain only a single uncleocapsic per viral envelope. The twelve isolates were genotypically characterized using restriction endonulcleases, Hind III, Eco RI, Sal I and Bam HI. Five distinct genotypic variants were found. The genotypic analysis of the variants rev Baled homogeneity and stability of the clones upon passage in vivo or in vitro. The isolation of the variants demonstrated the heterogeneity of the original uncloned virus population and the ability to clone MNPVs by plaque purification of extracellular nonoccluded virions. The cloning and genotypic analyses are also si gnificant with regard to understanding the genetic nature of multiply embedded NPVs, since each cloned virus gives rise to multiply-embedded virions; the DNA genome within each multiply embedded virion being identical genotypically.
The AcNPV L-1 clone was used as a wild type parental virus for mutagenesis with 5-bromodeoxyuridine -and N-methyl-N´-nitro-N-nitrosoguanidine. Sixteen temperature-sensitive conditional lethal mutants of AcNPV were isolated and initially characterized. Complementation analyses. performed by either measuring the increase in extracellular nonoccluded virus formation or by observing occluded virus formation in mixed infection at 32.5℃, demoastrated the presence of fifteen complementation groups. Recombination between two mutant viruses was observed and the recombination frequency was high. Some of properties of the is mutants were observed upon plaque assay at the nonpermissive temperature of 33.5℃. A Large proportion of the mutants were unable to form polyhedral occlusion bodies at 32.5℃. One mutants formed palques in which the cells lacked polyhedra at 32.5℃. Another mutant type was defective in the production of progeny extracellular nonoccluded virus and produced a plaque consisting of only a single cell containing polyhedra at 32.5℃. One mutant was defective in plaque formation, progeny NOV formation at 32.5℃. Several mutants produced NOVs but failed to produce plaques or polphedra at 32.5℃. Other phenotypes were also distinguished. Four of the mutants were found to be host-dependent in their temperature-sensitivity for polyhedra formation.
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